Inflammation is a pathological condition of an abscess caused by foreign infectious agents (bacteria, fungi, virus, various kinds of allergens, etc.). For example, when foreign bacteria invade into and proliferate in a tissue, the leukocytes of the body recognize and actively attack the proliferating foreign bacteria, during which leukocytes die and bacteria are killed by the leukocytes. The dead leukocytes and bacterial lysates accumulate in the tissue, forming an abscess. The abscess formed by inflammation can be treated through anti-inflammation activity. Anti-inflammation activity refers to a process that reduces inflammation in which the proliferation of the foreign agent, such as bacteria, is inhibited with the aid of an anti-inflammatory agent, for example, an antibacterial agent, or in which macrophages are activated to digest and excrete the foreign materials accumulated in the abscess.
Inflammation refers to a biological protective response of tissues to harmful stimuli. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process for rehabilitating the cells or tissues on which organic lesion has been imposed by the invasion of the stimuli. Factors involved in these serial processes are local vascular tissues, various tissue cells of the body fluid, immune cells, etc. With advances of molecular biology, attempts have recently been made to understand inflammatory diseases at molecular levels. As a result, factors responsible for inflammatory diseases have been gradually revealed.
Cytokines and mediators which induce inflammation are regulated by nuclear factors. To quote an example, NF-κB (nuclear factor-kappa B) is a nuclear protein of the Rel gene family, and to date, seven members of the NF-κB subfamily have been identified. While in an inactivated state, NF-κB is located in the cytosol, complexed with the inhibitory protein IκB (inhibitory kappa B). A variety of extracellular signals including reactive oxygen, chemokines such as TNF-α (tumor necrosis factor-α), and LPS (lipopolysaccharide) activate the enzyme IκB kinase. In turn, the IκB kinase phosphorylates IκB, which results in dissociation of IκB from NF-κB. The NF-κB thus activated, a heterodimer composed of p50 and p65, is then translocated into the nucleus where it binds to specific sequences of DNA to promote the expression of the target genes, for example, genes responsible for inflammation, such as tumor necrosis factor, cyclooxygenase, etc. (Oh G T et al., Artherosclerosis, 159(1): 17-26, 2001; Epstein F H et al., The New England Journal of Medicine, 336(15): 1066-1071, 1997; Zhang W J et al., FASEB J, 15(130): 2423-2431, 2001; Denk A et al., J. Biol. Chem., 276(30): 28451-28458, 2001; Sahnoun Z et al., Physiology, 53(4): 315-339, 1998; Lindner V Pathobiology, 66(6): 311-320, 1998; Landry D B et al., Am. J. Pathol., 151(4): 1085-1095, 1997; Gerritsen M E et al., Am. J. Pathol., 147(2): p278-292, 1995).
Nitric oxide is biosynthesized endogenously by the oxidation of L-arginine in the presence of nitric oxide synthase (NOS), and is an inflammatory mediator acting as a host defense by damaging pathogenic DNA and as a regulatory molecule with homeostatic activities (Kou and Schroder, Annals of Surgery 221, 220-235, 1995). In the NOS family, iNOS (inducible nitric oxide synthase) is known to be closely correlated with the intracellular overproduction of NO. PGE2 (prostaglandin E2) and leukotriene are inflammatory mediators that are biosynthesized from arachidonic acid. PGE2 is produced by the cyclooxygenase-2 enzyme (COX-2) and is abundantly found in macrophages and monocytes. Macrophages are induced by inflammatory agents, such as LPS, to be activated.
Asthma is a disease characterized by hypersensitivity of the airways to a variety of stimuli and results in variable and recurring symptoms including wheezing, shortness of breath, coughing, etc. which are often reversible either spontaneously or with specific therapy. Most asthma is allergic with symptoms of chronic airway inflammation and bronchial hyperresponsiveness (Minoguchi K and Adachi M. Pathophysiology of asthma. In: Chemiack N S, Altose M D, Homma I, editors. Rehabilitation of the patient with respiratory disease. New York: McGraw-Hill, 1999, pp 97-104).
Asthma may be classified as extrinsic or intrinsic based on whether symptoms are precipitated by allergens (extrinsic) or not (intrinsic). Patients with extrinsic asthma test positive in a skin test for allergies and in a bronchial provocation test, and extrinsic asthma is usually developed from childhood. Dust and dust mites are prevalent among allergens. In addition, pollen, epithelia from animals, and fungi are causative of asthma. For intrinsic asthma, symptoms occur or worsen in the presence of upper respiratory tract infections, exercise, emotional instability, cold weather, and moisture changes, and is observed in adult patients. Further, there are chemical-induced asthma, exercise-induced asthma, and occupational asthma.
Generally, asthma is known as a chronic inflammatory disease which is developed as inflammatory cells, after proliferation, differentiation, and activation by interleukin-4, -5, and -13 produced by Th2 (T helper 2) lymphocytes, migrate and invade the airways and tissues around the airways (Elias J A, et al., J. Clin. Invest., 111, pp 291-297, 2003). In this case, activated inflammatory cells such as eosinophils, mast cells, alveolar macrophages, etc. release various inflammatory mediators (cysteinyl leukotrienes, prostaglandins, etc.), playing an important role in bronchial constriction (Maggi E, Immunotechnology, 3, pp 233-244, 1998; Pawankar R., Curr. Opin. Allergy Clin. Immunol., 1, pp 3-6, 2001; Barnes P J, et al., Pharmacol Rev., 50, pp 515-596, 1998).
Accordingly, because cytokines IL-4, IL-5, and IL-13 and immunoglobulin, which are involved in the activation of inflammatory cells, and cysteinyl leukotrienes secreted from the inflammatory cells such as eosinophils, are main factors causing asthma, extensive research has been done to develop drugs inhibitory of the production or biosynthesis of the factors.
Steroid agents are the most potent anti-inflammatory drugs developed thus far. However, the long-term use of steroids is accompanied by side effects. In the treatment of asthma, steroid agents exert surprising therapeutic effects to the extent of completely removing the symptoms the first time, but this is transient. Symptoms revive with the cessation of use of steroids and are exacerbated with the repetition of their use. Sides effects of steroids include the development of a round, puffy face, fluid collection, adrenal insufficiency, an increase in susceptibility to infections, the occurrence of neurological problems, exacerbation of cataracts, glaucoma, and gastric ulcers, a delay in wound healing, and reactivation of latent infections.
Studies have been focused on materials which have anti-inflammatory effects without side effects. Particularly, extracts from plants which are inhibitory of inflammation and are safely applicable to foods without causing side effects have attracted intensive scientific interest. For example, with the fact in mind that NF-κB, a regulator of a gene population responsible for atopic dermatitis, binds to specific genes of immune cells to promote the expression of inflammatory mediators, a research team from Hirosaki University, Japan, in collaboration with a team from Osaka University, Japan, developed an atopy therapeutic comprising an artificial DNA mimicking the gene to which NF-κB binds, which blocks the activity of NF-κB by tricking NF-κB into the artificial DNA mimic, and demonstrated the clinical effectiveness of the therapeutic. Novartis Pharma A. G. in Basel, Switzerland, developed and markets “Elidel” derived from pimecrolimus, as a therapeutic for atopic dermatitis, which is an ascomycin macrolactam derivative functioning to selectively inhibit the synthesis and release of cytokines responsible for inflammation. In addition, a research team led by Prof. H. W. Chang in the College of Pharmacy, Yeungnam University, found that an extract from Saururus chinensis (Lour.) Baill and Ailanthus altissima SWINGLE is therapeutically effective for asthma and allergies, and was reported to have agreed on a contract with Korea Pharma Co. Ltd on Jan. 19, 2006 for technical transfer at a royalty of 4% of sales with a prepayment of 150 million won (from a report on Jan. 22, 2006, of the Korean Pharmaceutical Association News).
Many therapeutics for allergic and inflammatory diseases have been developed to target cytokines and chemokines involved in the onset of the diseases. Among them are compositions comprising kiwi fruit extracts, fermented cactus extracts, and lactic acid bacteria. Nowhere is the use of a velvet apple extract in the therapy of inflammation and asthma found in the prior art.
Velvet apple, also called Mabolo or Kamagong, is a plant of the genus of ebony trees belonging to the Ebenaceae family. It is native to the Philippines. Velvet apple timber is extremely dense and hard and is famous for its dark color. Like many other very hard woods, it is sometimes called “iron wood.” Its edible fruit has a skin covered in a fine, velvety fur which is usually reddish-brown, and soft, creamy, pink flesh, with a taste and aroma comparable to fruit cream cheese (Benedikt Mandl, Jimmy Wales, Wikimedia Foundation, 2004). U.S. Patent Publication No. 20080199533 discloses the use of velvet apple fruits as a cosmetic material. In addition, velvet apple fruits are used as food materials. A recent report has described that a mixture of several chemicals separated from an ethylacetate extract of spontaneously dried velvet apple leaves have analgesic and anti-inflammatory effects as demonstrated in a mouse test (Ragasa C Y et al., Nat. Prod. Res. 23(13): 1252-1258, 2009), but did not elucidate the mechanism of anti-inflammation or anti-asthmatic effects.
We, the present inventors, made an experiment to discover the anti-inflammation mechanism of a velvet apple extract. Artificial inflammation-induced macrophages, when treated with a velvet apple extract, were found to inhibit the nuclear translocation of NF-κB whose expression is promoted in response to inflammatory stimuli, which in turn suppresses the production of NO and PGE2, the expression of iNOS and COX-2, and the release of IL-1β and TNF-α, and significantly down-regulates Th2-mediated IL-4 and IL-13 production. In addition, an ovalbumin-induced asthma mouse model test showed that a velvet apple extract inhibits the increase of eosinophils in bronchoalveolar lavage fluid and suppresses the secretion of immunoglobulins and chemokines in bronchoalveolar lavage fluid and blood. Also, the velvet apple extract is found to be almost free of cytotoxicity and therefore is useful as an active ingredient of a pharmaceutical composition for preventing or treating various inflammatory disorders, allergic diseases, or asthma, which leads to the present invention.